Chemical and Biological Evaluation of Novel 1H-Chromeno[3,2-c]pyridine Derivatives as MAO Inhibitors Endowed with Potential Anticancer Activity

About twenty molecules sharing 1H-chromeno[3,2-c]pyridine as the scaffold and differing in the degree of saturation of the pyridine ring, oxidation at C10, 1-phenylethynyl at C1 and 1H-indol-3-yl fragments at C10, as well as a few small substituents at C6 and C8, were synthesized starting from 1,2,3,4-tetrahydro-2-methylchromeno[3,2-c]pyridin-10-ones (1,2,3,4-THCP-10-ones, 1) or 2,3-dihydro-2-methyl-1H-chromeno[3,2-c]pyridines (2,3-DHPCs, 2). The newly synthesized compounds were tested as inhibitors of the human isoforms of monoamine oxidase (MAO A and B) and cholinesterase (AChE and BChE), and the following main SARs were inferred: (i) The 2,3-DHCP derivatives 2 inhibit MAO A (IC50 about 1 μM) preferentially; (ii) the 1,2,3,4-THCP-10-one 3a, bearing the phenylethynyl fragment at C1, returned as a potent MAO B inhibitor (IC50 0.51 μM) and moderate inhibitor of both ChEs (IC50s 7–8 μM); (iii) the 1H-indol-3-yl fragment at C10 slightly increases the MAO B inhibition potency, with the analog 6c achieving MAO B IC50 of 3.51 μM. The MAO B inhibitor 3a deserves further pharmacological studies as a remedy in the symptomatic treatment of Parkinson’s disease and neuroprotectant for Alzheimer’s disease. Besides the established neuroprotective effects of MAO inhibitors, the role of MAOs in tumor insurgence and progression has been recently reported. Herein, antiproliferative assays with breast (MCF-7), colon (HCT116) and cisplatin-resistant ovarian (SK-OV-3) tumor cells revealed that the 10-indolyl-bearing 2,3,4,10-THCP analog 6c exerts anti-tumor activity with IC50s in the range 4.83–11.3 μM.

In this study, the introduction of X-substituted ethinyl fragments onto the tetrahydropyridine cycle expanded the synthetic and biological capabilities of the chromenopyridines. 1,2-Dihydrochromeno [3,2-c]pyridin-10-one (DHCP-10-one) derivatives 3a-d were obtained by a cross-coupling reaction of 1a-d with terminal alkynes in the presence of CuI and diisopropylazadicarboxylate (DIAD) (Scheme 2).
The reaction was conducted in dry THF, according to the previously reported synthetic procedure [8]. Most likely, in the first stage, the dihydropyridine cycle is oxidized under the action of DIAD to the pyridinium salt B, which is then alkynylated with copper acetylenide formed from the corresponding alkyne and CuI (Scheme 3). The molecular structure of 3a was confirmed by X-ray diffraction analysis (Figure 1). The reaction was conducted in dry THF, according to the previously reported synthetic procedure [8]. Most likely, in the first stage, the dihydropyridine cycle is oxidized under the action of DIAD to the pyridinium salt B, which is then alkynylated with copper acetylenide formed from the corresponding alkyne and CuI (Scheme 3).
The reaction was conducted in dry THF, according to the previously reported synthetic procedure [8]. Most likely, in the first stage, the dihydropyridine cycle is oxidized under the action of DIAD to the pyridinium salt B, which is then alkynylated with copper acetylenide formed from the corresponding alkyne and CuI (Scheme 3). The molecular structure of 3a was confirmed by X-ray diffraction analysis ( Figure 1). The molecular structure of 3a was confirmed by X-ray diffraction analysis ( Figure 1). 2,3-Dihydrochromeno [3,2-c]pyridines (2a and 2b) were functionalized with various nucleophiles under acid catalysis conditions. N-methyl pyrrole, nitromethane and indole or 5-substituted indoles were used as C-H nucleophiles. The reactions proceeded in CF 3 CH 2 OH under microwave-activation conditions. All nucleophilic addition products were isolated by column chromatography (Scheme 4).

Figure 1.
Crystal structure of compound 3a (CCDC 2225696). All atoms are color-labeled as red (oxygen), blue (nitrogen), gray (carbon), white (hydrogen). (see Table S1 for the crystal data and structure refinement for 3a). [3,2-c]pyridines (2a and 2b) were functionalized with various nucleophiles under acid catalysis conditions. N-methyl pyrrole, nitromethane and indole or 5-substituted indoles were used as C-H nucleophiles. The reactions proceeded in CF3CH2OH under microwave-activation conditions. All nucleophilic addition products were isolated by column chromatography (Scheme 4). We supposed that the reaction proceeds through the acid-catalyzed transformation of the DHCP cycle to benzopyryl salts. The latter readily takes part in the reactions of nucleophilic addition. The probable mechanism of these transformations is shown in Scheme 5.   Table S1 for the crystal data and structure refinement for 3a).  Table S1 for the crystal data and structure refinement for 3a).

2,3-Dihydrochromeno
2,3-Dihydrochromeno [3,2-c]pyridines (2a and 2b) were functionalized with various nucleophiles under acid catalysis conditions. N-methyl pyrrole, nitromethane and indole or 5-substituted indoles were used as C-H nucleophiles. The reactions proceeded in CF3CH2OH under microwave-activation conditions. All nucleophilic addition products were isolated by column chromatography (Scheme 4). We supposed that the reaction proceeds through the acid-catalyzed transformation of the DHCP cycle to benzopyryl salts. The latter readily takes part in the reactions of nucleophilic addition. The probable mechanism of these transformations is shown in Scheme 5. We supposed that the reaction proceeds through the acid-catalyzed transformation of the DHCP cycle to benzopyryl salts. The latter readily takes part in the reactions of nucleophilic addition. The probable mechanism of these transformations is shown in Scheme 5. Recently, some of us proved that 12H-chromeno [2,3-c]isoquinoline bearing the 1Hindolyl group at C12 has antiproliferative activity against diverse human tumor cells (i.e., MCF-7, HCT116, A2780, SK-OV-3) with IC50s in the low micromolar range [9]. Due to a certain degree of molecular similarity with that compound of the indole-bearing THCP derivatives 6a-c synthesized in this study, we tried to optimize a simpler procedure for their synthesis. Earlier the synthesis of similar compounds had been accomplished through multicomponent reactions of salicylic aldehydes with C-H acids and various nucleophiles [10][11][12]. The key factors for the reactions' success were the catalytic system, temperature, and the type of process activation.
The interaction of salicylic aldehyde, 4-methylpiperidine and 1H-indole was performed in EtOH under heating in the presence of 10 mol% L-proline. However, instead of the expected product 6, hemiacetal 7 was obtained as a white crystalline substance (Scheme 6). We tried to optimize the reaction conditions by varying solvents, temperatures and catalysts (Table 1). The expected product 6 was obtained in just 12% yield only in CF3CH2OH as the solvent.  Recently, some of us proved that 12H-chromeno [2,3-c]isoquinoline bearing the 1Hindolyl group at C12 has antiproliferative activity against diverse human tumor cells (i.e., MCF-7, HCT116, A2780, SK-OV-3) with IC 50 s in the low micromolar range [9]. Due to a certain degree of molecular similarity with that compound of the indole-bearing THCP derivatives 6a-c synthesized in this study, we tried to optimize a simpler procedure for their synthesis. Earlier the synthesis of similar compounds had been accomplished through multicomponent reactions of salicylic aldehydes with C-H acids and various nucleophiles [10][11][12]. The key factors for the reactions' success were the catalytic system, temperature, and the type of process activation.
The interaction of salicylic aldehyde, 4-methylpiperidine and 1H-indole was performed in EtOH under heating in the presence of 10 mol% L-proline. However, instead of the expected product 6, hemiacetal 7 was obtained as a white crystalline substance (Scheme 6). We tried to optimize the reaction conditions by varying solvents, temperatures and catalysts (Table 1). The expected product 6 was obtained in just 12% yield only in CF 3 CH 2 OH as the solvent. Recently, some of us proved that 12H-chromeno [2,3-c]isoquinoline bearing the 1Hindolyl group at C12 has antiproliferative activity against diverse human tumor cells (i.e., MCF-7, HCT116, A2780, SK-OV-3) with IC50s in the low micromolar range [9]. Due to a certain degree of molecular similarity with that compound of the indole-bearing THCP derivatives 6a-c synthesized in this study, we tried to optimize a simpler procedure for their synthesis. Earlier the synthesis of similar compounds had been accomplished through multicomponent reactions of salicylic aldehydes with C-H acids and various nucleophiles [10][11][12]. The key factors for the reactions' success were the catalytic system, temperature, and the type of process activation.
The interaction of salicylic aldehyde, 4-methylpiperidine and 1H-indole was performed in EtOH under heating in the presence of 10 mol% L-proline. However, instead of the expected product 6, hemiacetal 7 was obtained as a white crystalline substance (Scheme 6). We tried to optimize the reaction conditions by varying solvents, temperatures and catalysts (Table 1). The expected product 6 was obtained in just 12% yield only in CF3CH2OH as the solvent.  Recently, some of us proved that 12H-chromeno [2,3-c]isoquinoline bearing the 1Hindolyl group at C12 has antiproliferative activity against diverse human tumor cells (i.e., MCF-7, HCT116, A2780, SK-OV-3) with IC50s in the low micromolar range [9]. Due to a certain degree of molecular similarity with that compound of the indole-bearing THCP derivatives 6a-c synthesized in this study, we tried to optimize a simpler procedure for their synthesis. Earlier the synthesis of similar compounds had been accomplished through multicomponent reactions of salicylic aldehydes with C-H acids and various nucleophiles [10][11][12]. The key factors for the reactions' success were the catalytic system, temperature, and the type of process activation.
The interaction of salicylic aldehyde, 4-methylpiperidine and 1H-indole was performed in EtOH under heating in the presence of 10 mol% L-proline. However, instead of the expected product 6, hemiacetal 7 was obtained as a white crystalline substance (Scheme 6). We tried to optimize the reaction conditions by varying solvents, temperatures and catalysts (Table 1). The expected product 6 was obtained in just 12% yield only in CF3CH2OH as the solvent.  Recently, some of us proved that 12H-chromeno [2,3-c]isoquinoline bearing the 1Hindolyl group at C12 has antiproliferative activity against diverse human tumor cells (i.e., MCF-7, HCT116, A2780, SK-OV-3) with IC50s in the low micromolar range [9]. Due to a certain degree of molecular similarity with that compound of the indole-bearing THCP derivatives 6a-c synthesized in this study, we tried to optimize a simpler procedure for their synthesis. Earlier the synthesis of similar compounds had been accomplished through multicomponent reactions of salicylic aldehydes with C-H acids and various nucleophiles [10][11][12]. The key factors for the reactions' success were the catalytic system, temperature, and the type of process activation.
The interaction of salicylic aldehyde, 4-methylpiperidine and 1H-indole was performed in EtOH under heating in the presence of 10 mol% L-proline. However, instead of the expected product 6, hemiacetal 7 was obtained as a white crystalline substance (Scheme 6). We tried to optimize the reaction conditions by varying solvents, temperatures and catalysts (Table 1). The expected product 6 was obtained in just 12% yield only in CF3CH2OH as the solvent.  While we failed in finding the conditions for the three-component synthesis of 6a, it is worth highlighting that the resulting cyclic hemiacetal 7a has not been described anywhere before and it was of interest as a new derivative of the chromene series. Keeping this in mind, we obtained a series of 7a-d hemiacetals by reactions of salicylic aldehyde and its substituted derivatives with N-methylpyrrolidone in EtOH in the presence of L-proline as the catalyst. The products were crystallized and isolated by filtration (Scheme 7). While we failed in finding the conditions for the three-component synthesis of 6a, it is worth highlighting that the resulting cyclic hemiacetal 7a has not been described anywhere before and it was of interest as a new derivative of the chromene series. Keeping this in mind, we obtained a series of 7a-d hemiacetals by reactions of salicylic aldehyde and its substituted derivatives with N-methylpyrrolidone in EtOH in the presence of L-proline as the catalyst. The products were crystallized and isolated by filtration (Scheme 7).

Scheme 7. Synthesis of hemiacetals 7a-f.
The structure of the cyclic hemiacetal 7c was confirmed by X-ray diffraction data (Figure 2), which allowed us to establish that two hydroxyl groups of the central cycle have relative trans-configuration, whereas the azadecalin system of fused two non-aromatic six-membered cycles has a cis-configuration ( Figure 3). In this way, the compound having 4aR*, 10S*, 10aR* configuration of asymmetric centers is formed in the reaction diastereoselectively.

L-proline
EtOH The structure of the cyclic hemiacetal 7c was confirmed by X-ray diffraction data (Figure 2), which allowed us to establish that two hydroxyl groups of the central cycle have relative trans-configuration, whereas the azadecalin system of fused two non-aromatic six-membered cycles has a cis-configuration ( Figure 3). In this way, the compound having 4aR*, 10S*, 10aR* configuration of asymmetric centers is formed in the reaction diastereoselectively. While we failed in finding the conditions for the three-component synthesis of 6a, it is worth highlighting that the resulting cyclic hemiacetal 7a has not been described anywhere before and it was of interest as a new derivative of the chromene series. Keeping this in mind, we obtained a series of 7a-d hemiacetals by reactions of salicylic aldehyde and its substituted derivatives with N-methylpyrrolidone in EtOH in the presence of L-proline as the catalyst. The products were crystallized and isolated by filtration (Scheme 7).

Scheme 7. Synthesis of hemiacetals 7a-f.
The structure of the cyclic hemiacetal 7c was confirmed by X-ray diffraction data (Figure 2), which allowed us to establish that two hydroxyl groups of the central cycle have relative trans-configuration, whereas the azadecalin system of fused two non-aromatic six-membered cycles has a cis-configuration ( Figure 3). In this way, the compound having 4aR*, 10S*, 10aR* configuration of asymmetric centers is formed in the reaction diastereoselectively.  Table S2 for the crystal data and structure refinement for 7c).

L-proline
EtOH  Table S2 for the crystal data and structure refinement for 7c).
It should be noted that according to the X-ray data, compound 7c is formed as a racemate (the spatial symmetry group of the formed crystal P21/C, i.e., centrosymmetric). This means the co-crystallization of both enantiomers in one crystal. It should be noted that according to the X-ray data, compound 7c is formed as a racemate (the spatial symmetry group of the formed crystal P21/C, i.e., centrosymmetric). This means the co-crystallization of both enantiomers in one crystal.
Probably, the first stage of the mechanism of hemiacetals 7a-f formation involves the reaction of a salicylic aldehyde with L-proline yielding the intermediate A. The latter is nucleophilically attacked by the enol form of piperidone to form intermediate B, the conformation of which is fixed by the formation of HB between the protonated carbonyl group of piperidone and the carboxylate group of proline. Thus, free rotation around the bond between carbon in α-position of the benzene ring and piperidine fragment (marked on the scheme) is blocked yielding cis-decalin system of the cyclic hemiacetals 7 (Scheme 8). To confirm the role of L-proline in the stereoselective formation of 7, we performed reactions of salicylic aldehydes with 4-methylpiperidone without its addition. As a result, a series of compounds 8a-e was obtained with almost the same yields as for reactions in the presence of proline (Scheme 9).
However, the stereochemistry of compound 8 differs from the stereochemistry of compound 7 (Figure 3). The two hydroxyl groups of the central cycle have the same configuration as in the case of compound 8a, but the fusion of six-membered cycles of the azadecalin   It should be noted that according to the X-ray data, compound 7c is formed as a racemate (the spatial symmetry group of the formed crystal P21/C, i.e., centrosymmetric). This means the co-crystallization of both enantiomers in one crystal.
Probably, the first stage of the mechanism of hemiacetals 7a-f formation involves the reaction of a salicylic aldehyde with L-proline yielding the intermediate A. The latter is nucleophilically attacked by the enol form of piperidone to form intermediate B, the conformation of which is fixed by the formation of HB between the protonated carbonyl group of piperidone and the carboxylate group of proline. Thus, free rotation around the bond between carbon in α-position of the benzene ring and piperidine fragment (marked on the scheme) is blocked yielding cis-decalin system of the cyclic hemiacetals 7 (Scheme 8). To confirm the role of L-proline in the stereoselective formation of 7, we performed reactions of salicylic aldehydes with 4-methylpiperidone without its addition. As a result, a series of compounds 8a-e was obtained with almost the same yields as for reactions in the presence of proline (Scheme 9).
However, the stereochemistry of compound 8 differs from the stereochemistry of compound 7 (Figure 3). The two hydroxyl groups of the central cycle have the same configuration as in the case of compound 8a, but the fusion of six-membered cycles of the azadecalin To confirm the role of L-proline in the stereoselective formation of 7, we performed reactions of salicylic aldehydes with 4-methylpiperidone without its addition. As a result, a series of compounds 8a-e was obtained with almost the same yields as for reactions in the presence of proline (Scheme 9).
However, the stereochemistry of compound 8 differs from the stereochemistry of compound 7 (Figure 3). The two hydroxyl groups of the central cycle have the same configuration as in the case of compound 8a, but the fusion of six-membered cycles of the azadecalin system has trans-configuration ( Figure 4). Thus, the reaction diastereoselectively yields compounds with a relative 4aR*,10S*,10aS* configuration of asymmetric centers. system has trans-configuration ( Figure 4). Thus, the reaction diastereoselectively yields compounds with a relative 4aR*,10S*,10aS* configuration of asymmetric centers.   Table S3 for the crystal data and structure refinement for 8a).
It is worth mentioning that the 1 H NMR spectra of the isomeric 7 and 8 are very close having similar chemical shifts of protons signals and spin-spin coupling constants (the difference in chemical shifts of the corresponding protons is no more than 0.01 ppm). However, these compounds have different melting points (values in Table 2). system has trans-configuration ( Figure 4). Thus, the reaction diastereoselectively yields compounds with a relative 4aR*,10S*,10aS* configuration of asymmetric centers.  All atoms are color-labeled as red (oxygen), blue (nitrogen), gray (carbon). (see Table S3 for the crystal data and structure refinement for 8a).
It is worth mentioning that the 1 H NMR spectra of the isomeric 7 and 8 are very close having similar chemical shifts of protons signals and spin-spin coupling constants (the difference in chemical shifts of the corresponding protons is no more than 0.01 ppm). However, these compounds have different melting points (values in Table 2).  All atoms are color-labeled as red (oxygen), blue (nitrogen), gray (carbon). (see Table S3 for the crystal data and structure refinement for 8a).
It is worth mentioning that the 1 H NMR spectra of the isomeric 7 and 8 are very close having similar chemical shifts of protons signals and spin-spin coupling constants (the difference in chemical shifts of the corresponding protons is no more than 0.01 ppm). However, these compounds have different melting points (values in Table 2). system has trans-configuration ( Figure 4). Thus, the reaction diastereoselectively yields compounds with a relative 4aR*,10S*,10aS* configuration of asymmetric centers.  All atoms are color-labeled as red (oxygen), blue (nitrogen), gray (carbon). (see Table S3 for the crystal data and structure refinement for 8a).
It is worth mentioning that the 1 H NMR spectra of the isomeric 7 and 8 are very close having similar chemical shifts of protons signals and spin-spin coupling constants (the difference in chemical shifts of the corresponding protons is no more than 0.01 ppm). However, these compounds have different melting points (values in Table 2). system has trans-configuration ( Figure 4). Thus, the reaction diastereoselectively yields compounds with a relative 4aR*,10S*,10aS* configuration of asymmetric centers.  All atoms are color-labeled as red (oxygen), blue (nitrogen), gray (carbon). (see Table S3 for the crystal data and structure refinement for 8a).
It is worth mentioning that the 1 H NMR spectra of the isomeric 7 and 8 are very close having similar chemical shifts of protons signals and spin-spin coupling constants (the difference in chemical shifts of the corresponding protons is no more than 0.01 ppm). However, these compounds have different melting points (values in Table 2). system has trans-configuration ( Figure 4). Thus, the reaction diastereoselectively yields compounds with a relative 4aR*,10S*,10aS* configuration of asymmetric centers.  All atoms are color-labeled as red (oxygen), blue (nitrogen), gray (carbon). (see Table S3 for the crystal data and structure refinement for 8a).
It is worth mentioning that the 1 H NMR spectra of the isomeric 7 and 8 are very close having similar chemical shifts of protons signals and spin-spin coupling constants (the difference in chemical shifts of the corresponding protons is no more than 0.01 ppm). However, these compounds have different melting points (values in Table 2). Such diastereoselectivity in the synthesis of hemiacetals 8 could be explained by the formation of the more thermodynamically preferable trans-decalin system due to the possibility of the attack of phenolic hydroxyl from both possible sides in intermediates B and C (Scheme 10).
Such diastereoselectivity in the synthesis of hemiacetals 8 could be explained by the formation of the more thermodynamically preferable trans-decalin system due to the possibility of the attack of phenolic hydroxyl from both possible sides in intermediates B and C (Scheme 10). Such diastereoselectivity in the synthesis of hemiacetals 8 could be explained by the formation of the more thermodynamically preferable trans-decalin system due to the possibility of the attack of phenolic hydroxyl from both possible sides in intermediates B and C (Scheme 10).

Scheme 10.
Mechanism of the synthesis of compounds 8.

Inhibition of Monoamine Oxidases and Cholinesterases
Diverse 1H-chromeno [3,2-c]pyridine derivatives synthesized herein, with different saturation degree of the pyridine ring and substituents (R, R 1 , R 2 and X), were tested as inhibitors of the human isoforms of MAO (A and B) and ChE (AChE and BChE). To extend the knowledge of the structure-activity relationships (SARs) of this class of annulated oxaza-heterocyclic derivatives, the activities of the previously reported 1,2,3,4-tetrahydrochromeno [3,2-c]pyridin-10-one (1,2,3,4-THCP-10-one) 1a were evaluated and compared with those of 2,3-dihydro-1H-chromeno [3,2-c] All of the compounds were tested against each enzyme at a single 10 μM concentration, and for those showing more than 60% inhibition at that concentration, IC50s were determined in at least three independent experiments (Table 3).
Except for the 1-(2-phenylethynyl) derivative of 1,2-DHCP 3a, which achieved singledigit micromolar IC50 values against both ChEs, all the newly synthesized chromeno [3,2c]pyridine derivatives resulted scarcely active as ChEs inhibitors. 2,3-DHCPs 2a and 2b returned good MAO A inhibition data (IC50s 1.18 and 0.703 μM, respectively) and about tenfold selectivity over MAO B, whereas the MAO inhibitory activity of the 1,2-DHCP-10ones 3a-c was affected by the substituents at N2 and C1. Within the limits of the explored property space around the 1,2-DHCP-10-one scaffold, the pattern 2-methyl/1-phenylethynyl (3a and 3b) solely warranted submicromolar MAO B inhibition and more than twentyfold MAO B/A selectivity, while bulkier substituents at N2 caused a drop of the inhibitory potency. These results are congruent with those obtained from a very recent

Inhibition of Monoamine Oxidases and Cholinesterases
Diverse 1H-chromeno [3,2-c]pyridine derivatives synthesized herein, with different saturation degree of the pyridine ring and substituents (R, R 1 , R 2 and X), were tested as inhibitors of the human isoforms of MAO (A and B) and ChE (AChE and BChE). To extend the knowledge of the structure-activity relationships (SARs) of this class of annulated oxaza-heterocyclic derivatives, the activities of the previously reported 1,2,3,4tetrahydrochromeno [3,2-c]pyridin-10-one (1,2,3,4-THCP-10-one) 1a were evaluated and compared with those of 2,3-dihydro-1H-chromeno [3,2-c] All of the compounds were tested against each enzyme at a single 10 µM concentration, and for those showing more than 60% inhibition at that concentration, IC 50 s were determined in at least three independent experiments (Table 3). Except for the 1-(2-phenylethynyl) derivative of 1,2-DHCP 3a, which achieved singledigit micromolar IC 50 values against both ChEs, all the newly synthesized chromeno [3,2c]pyridine derivatives resulted scarcely active as ChEs inhibitors. 2,3-DHCPs 2a and 2b returned good MAO A inhibition data (IC 50 s 1.18 and 0.703 µM, respectively) and about tenfold selectivity over MAO B, whereas the MAO inhibitory activity of the 1,2-DHCP-10-ones 3a-c was affected by the substituents at N2 and C1. Within the limits of the explored property space around the 1,2-DHCP-10-one scaffold, the pattern 2-methyl/1phenylethynyl (3a and 3b) solely warranted submicromolar MAO B inhibition and more than twentyfold MAO B/A selectivity, while bulkier substituents at N2 caused a drop of the inhibitory potency. These results are congruent with those obtained from a very recent exploration of the 1,2,3,4-tetrahydrobenzo[b][1,6]naphthyridine scaffold of novel MAO inhibitors [13]. Indeed, the 1-(2-(4-fluorophenyl)ethynyl)-2-methyl analog proved to be in vitro a potent MAO B inhibitor with IC 50 of 1.35 µM. Comparing the MAO B inhibition data of 3a-b with those of 3c-d, and 1a as well, it appears that (i) the phenylethynyl group at C1 may be accommodated into the MAO B binding site better than into that of MAO A, (ii) the presence of the OMe or OEt substituents at C6 does not affect the MAO B inhibition, whereas (iii) alkyls (Et, iPr) bulkier than the Me group on N2 cause at least a twentyfold decrease in MAO B inhibition potency.
We sampled a few hemiacetals, three with cis-ring fusion (7a,c,e) and three with trans-ring fusion (8a,c,e), and assayed them for MAOs and ChEs inhibitory activities (Table 4). Regardless, the ring fusion stereochemistry (4aR,10S,10aR for compounds 7 and 4aR,10S,10aS for compounds 8), position and lipophilicity of the substituents, all the cyclic hemiacetals resulted poor inhibitors of MAO B with IC 50 s > 10 µM and in most cases even poorer inhibitors of AChE. The inhibitory activity of all the tested hemiacetals toward MAO A and BChE at 10 µM concentration was found to be very weak or null. The investigation of the inhibition kinetics of 3b and 6c, taken as representative of the two subsets of MAO B-selective inhibitors, resulted in Michaelis-Menten curves' fitting for competitive MAO B inhibition ( Figure 5), with inhibition constant (K i ) values equal to 1.41 ± 0.21 µM and 6.47 ± 0.22 µM, respectively.
The investigation of the inhibition kinetics of 3b and 6c, taken as representative of the two subsets of MAO B-selective inhibitors, resulted in Michaelis-Menten curves' fitting for competitive MAO B inhibition ( Figure 5), with inhibition constant (Ki) values equal to 1.41 ± 0.21 μM and 6.47 ± 0.22 μM, respectively.

Antiproliferative Activity on Tumor Cell Lines
Some years ago, some of us investigated the antiproliferative activity of 12Hchromeno [2,3-c]isoquinoline derivatives [9], among which the analog bearing 1H-indol-3yl moiety at C12 achieved a 50% inhibition of cell growth in some tumor cell lines, including the cisplatin-resistant ovarian carcinoma one, in the low micromolar range. Considering the molecular similarity of that compound with 10-(1H-indol-3-yl)-bearing 2,3,4,10-THCP analogs (6), we assayed compounds 6a-c, along with 2a-b and 3b-c, in three human tumor cell lines, i.e., breast (MCF-7), colon (HCT116) and ovarian resistant (SK-OV-3) tumor cells. Cisplatin (CDDP) and doxorubicin (DXR) were used as positive controls. Besides the antitumor activity, we were also interested in challenging recent studies supporting the role of monoamine oxidases in tumor proliferation [14]. Such evidence would give new chances to MAO inhibitors for being repositioned as coadjutants in the chemotherapy of drug-resistant tumors. In this light, disclosing compounds with multitarget activity, combining MAO inhibition and antiproliferative effects, would strengthen the validity of the multitargeting approach in anticancer therapy.
The cytotoxicity data in Table 5 indicate that 10-indolyl THCP analogs 6a-c are the most active among the tested compounds. They showed selectivity towards the MCF-7 line, with single-digit micromolar IC50s (4.80  6.82 μM). Compounds 2a-b and 3b-c resulted in lower antitumor activity in MCF-7 cell line and poorly active at 50 μM concentration in SK-OV-3 cells.

Antiproliferative Activity on Tumor Cell Lines
Some years ago, some of us investigated the antiproliferative activity of 12H-chromeno isoquinoline derivatives [9], among which the analog bearing 1H-indol-3-yl moiety at C12 achieved a 50% inhibition of cell growth in some tumor cell lines, including the cisplatin-resistant ovarian carcinoma one, in the low micromolar range. Considering the molecular similarity of that compound with 10-(1H-indol-3-yl)-bearing 2,3,4,10-THCP analogs (6), we assayed compounds 6a-c, along with 2a-b and 3b-c, in three human tumor cell lines, i.e., breast (MCF-7), colon (HCT116) and ovarian resistant (SK-OV-3) tumor cells. Cisplatin (CDDP) and doxorubicin (DXR) were used as positive controls. Besides the antitumor activity, we were also interested in challenging recent studies supporting the role of monoamine oxidases in tumor proliferation [14]. Such evidence would give new chances to MAO inhibitors for being repositioned as coadjutants in the chemotherapy of drug-resistant tumors. In this light, disclosing compounds with multitarget activity, combining MAO inhibition and antiproliferative effects, would strengthen the validity of the multitargeting approach in anticancer therapy.
The cytotoxicity data in Table 5 indicate that 10-indolyl THCP analogs 6a-c are the most active among the tested compounds. They showed selectivity towards the MCF-7 line, with single-digit micromolar IC 50 s (4.80 ÷ 6.82 µM). Compounds 2a-b and 3b-c resulted in lower antitumor activity in MCF-7 cell line and poorly active at 50 µM concentration in SK-OV-3 cells. Spearman's rank analysis [15] for the pairs 6a/CDDP/DXR, 6b/CDDP/DXR and 6c/CDDP/DXR provided very low values of Spearman indexes (ρ = −0.5), suggesting that the growth inhibition profile of the most active 6a-c in all the examined tumor cell lines is different from both CDDP and DXR. Considering that the SK-OV-3 cell line is characterized by intrinsic resistance, the antiproliferative activity of compounds 6a-c led us to hypothesize their use towards resistant lines and/or a synergistic action with drugs used in conventional therapies.

Structure-Activity Relationships
The biological evaluation of the newly synthesized molecules provided us with SARs which may helpfully suggest and support future 'hit-to-lead' molecular optimization studies of 1H-chromeno [3,2-c]pyridine analogs as MAO isoform-selective inhibitors or MAO-targeted neuroprotectant MTDLs, in combination with molecular modeling results obtained by others on chromone-based natural and synthetic compounds [2,16]. From the SAR perspective, within the limits of the biological and physicochemical space explored, this 'target-to-hit' study proves that: (i) The 2,3-DHCP derivatives 2a-b inhibit preferentially MAO A with IC 50 s of about 1 µM; (ii) the most potent MAO B-selective inhibitors are the 2-methyl 1,2,3,4-THCP-10-one derivatives 3a and 3b (IC 50 s 0.51 and 0.63 µM), which bear a phenylethynyl fragment at C1; 3a achieved also IC 50 s of 7-8 µM against both ChEs; (iii) installing the 1H-indol-3-yl fragment on C10 of the starting compound 2a did slightly improve the MAO B inhibition potency, and AChE as well, with a small effect of the 5-Brindolyl on the activity of 6c, which inhibited MAO B with a potency (IC 50 3.51 µM) close to that of pargyline; (iv) irrespective of their diastereisomerism, the cyclic hemiacetals 7 and 8 lose inhibition potency against the tested enzymes, likely owing to a loss of flatness compared to their more closely related compounds 2.
In addition, the tumor growth inhibitory activity assayed in three cell lines (i.e., MCF-7, HCT116 and SK-OV-3) suggests that the 10-(1H-indol-3-yl)-bearing 2,3,4,10-THCP analogs 6a-c are noteworthy. Based on molecular docking models and fluorescence quenching experiments, carried out by some of us on similar molecules [9], a propensity of compound 6 to bind DNA cannot be ruled out. However, 6c exerted antiproliferative effects with IC 50 s < 10 µM, with a value of 11 µM measured toward the cisplatin-resistant ovarian tumor cells (SK-OV-3). Alongside a more in-depth mechanistic investigation and molecular optimization, the hit compound 6c would also deserve to be tested against other tumor cell lines, trying to improve its delivery through suitable formulations [17]. . IR spectra were recorded on an Infralum FT-801 FTIR spectrometer (ISP SB RAS, Novosibirsk, Russia). The samples were analyzed as KBr disk solids, and the most important frequencies in cm −1 are reported. 1 H and 13 C NMR spectra were recorded in chloroform-d 3 (CDCl 3 ) or dimethylsulfoxide-d 6 (DMSO-d 6 ) solutions at 25 • C, with a 600-MHz NMR spectrometer (JEOL Ltd., Tokyo, Japan). Peak positions were given in parts per million (ppm) referenced to the appropriate solvent residual peak, and signal multiplicities were collected by: s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), ddd (doublet of doublet of doublet), tt (triplet of triplets), br.s (broad singlet) and m (multiplet). MALDI mass spectra were recorded using a Bruker autoflex speed instrument operating in positive reflectron mode (Bremen, Germany). The data of 3a, 7c and 8a were collected at room temperature using a STOE diffractometer Pilatus100K detector, focusing on mirror collimation Cu Kα (1.54086 Å) radiation, in rotation method mode. STOE X-AREA software was used for cell refinement and data reduction. Data collection and image processing were performed with X-Area 1.67 reported protocols [18,19]. The AChE activity was determined in an assay solution containing AChE (0.09 U/mL), 5,5 -dithiobis(2-nitrobenzoic acid) (i.e., the Ellman's reagent, 0.33 mM), the test compound (10 µM concentration, or seven scalar concentrations for compounds achieving > 60% enzyme inhibition at 10 µM), in 0.1 M PBS pH 8.0. After 20 min incubation at 25 • C, the substrate acetylthiocholine iodide (5 µM) was added, and its hydrolysis rates were monitored for 5.0 min at 412 nm. The BChE inhibitory activity was similarly determined by using BChE (0.09 U/mL) and butyrylthiocholine iodide (5 µM) as the substrate. IC 50 value, determined by the nonlinear regression method 'log[inhibitor] vs. response', or the % inhibition at 10 µM, is expressed as the mean ± SD of three independent measurements, each one performed in duplicate.

Materials and Methods
The IC 50 values, Michaelis-Menten curve fitting and inhibition constant (K i ) were calculated by nonlinear regression, using Prism software.

Cell Viability Assays
The SK-OV-3 ovarian cancer cell line, MCF-7 breast cancer line and HCT-116 colon cancer cell line were obtained from the National Cancer Institute, Biological Testing Branch (Frederick, MD, USA), and maintained in the logarithmic phase at 37 • C in a 5% CO 2 humidified air in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM glutamine, penicillin (100 U/mL) and streptomycin (0.1 mg/mL).
The growth inhibitory effects of compounds under investigation were compared to those of cisplatin (CDDP) and doxorubicin (DXR), used as positive controls, and evaluated by using the sulforhodamine-B (SRB) assay [20]. Briefly, cells were seeded into 96-well microtiter plates in 100 µL of the appropriate culture medium at plating densities at 2500, 5000 and 8000 cells/well for MCF-7, HCT-116 and SKOV-3, respectively, depending upon the doubling time of individual cell lines. After seeding, microtiter plates were incubated at 37 • C for 24 h before adding the test compounds. After 24 h, several samples of each cell line were fixed in situ with cold trichloroacetic acid (TCA) to represent a measurement of the cell population at the time of compound addition. The test compounds were freshly dissolved in dimethyl sulfoxide (DMSO, 10 −2 M) and gradually diluted to different concentrations (0.79-50 µM) with a complete medium, so that the maximum DMSO/well ratio was 0.5% v/v. After the addition of different compound concentrations to triplicate wells, the plates were further incubated at 37 • C for 72 h. Cells were fixed in situ by the gentle addition of 50 µL of cold 50% w/v TCA (final concentration 10%) and incubated for 1 h at 4 • C. The supernatant was discarded, and the plates were washed with tap water and air-dried. SRB solution (100 µL) at 0.4% (w/v) in 1% acetic acid was added to each well, and the plates were incubated for 30 min at room temperature. After staining, the unbound dye was removed by washing with 1% acetic acid and the plates were air-dried. The bound stain was then solubilized with 10mM Trizma base and the absorbance was read on an automatic plate reader at 570 nm. The compound concentration able to inhibit cell growth by 50% (IC 50 ± SD) was then calculated from semi-logarithmic dose-response plots.
Beyond some important SARs deduced from the biological evaluation of the newly synthesized compounds, all sharing a common 1H-chromeno [3,2-c]pyridine scaffold, potent MAO B inhibitors, namely 3a and 3b, were disclosed. Compound 3a, also endowed with moderate activity against AChE/BChE, is a hit deserving further pharmacological studies, as a possible remedy in early symptoms of PD [27] and/or as an anti-oxidative neuroprotectant in AD patients [28].
While the neuroprotective effects of MAO inhibitors in neurological diseases have long been studied, albeit with a low success rate in terms of clinical entries, the role of MAOs in tumor insurgence and progression has been only recently reported [29]. The inhibition of tumor cell growth, as assessed for several newly synthesized derivatives in antiproliferative assays with MCF-7, HCT116 and SK-OV-3 cell lines, suggest that the 10-(1H-indol-3-yl)-bearing 2,3,4,10-THCP analog 6c is noteworthy. Although these findings do not allow us to establish how much the inhibition of MAOs affects the antitumor effect, the combination of MAO inhibition with cytotoxicity toward tumor cells, such as that observed herein in 6a-c, might represent an approach worthy of study in cancer treatment.